Characterizing Different Modes of Interplay Between Rap1 and H3 Using Inducible H3-depletion Yeast.

Histones and transcription factors (TFs) are two important DNA-binding proteins that interact, compete, and together regulate transcriptional processes in response to diverse internal and external stimuli. Condition-specific depletion of histones in Saccharomyces cerevisiae using a galactose-inducible H3 promoter provides a suitable framework for examining transcriptional alteration resulting from reduced nucleosome content. However, the effect on DNA binding activities of TFs is yet to be fully explored. In this work, we combine ChIP-seq of H3 with RNA-seq to elucidate the genome-scale relationships between H3 occupancy patterns and transcriptional dynamics before and after global H3 depletion. ChIP-seq of Rap1 is also conducted in the H3-depletion and control treatments, to investigate the interplay between this master regulator TF and nucleosomal H3, and to explore the impact on diverse transcriptional responses of different groups of target genes and functions. Ultimately, we propose a working model and testable hypotheses regarding the impact of global and local H3 depletion on transcriptional changes. We also demonstrate different potential modes of interaction between Rap1 and H3, which sheds light on the potential multifunctional regulatory capabilities of Rap1 and potentially other pioneer factors.

Overexpression of Holocarboxylase Synthetase Predicts Lymph Node Metastasis and Unfavorable Prognosis in Breast Cancer.

BACKGROUND/AIM: Holocarboxylase synthetase (HLCS) catalyzes the specific attachment of biotin onto biotin-dependent carboxylases (BDCs) which play important roles in intermediary metabolism. Previous studies show that BDCs are overexpressed in many cancer types. However, expression of HLCS in cancerous tissues has not been reported. MATERIALS AND METHODS: Immunohistochemistry was used to investigate HLCS expression in breast tissue obtained from 65 Thai patients, and the correlation between its expression and key clinical-pathological parameters was assessed. The role of HLCS in supporting invasion was investigated in HLCS-knockdown MCF-7 cells. RESULTS: Overexpression of HLCS was significantly associated with metastasis of breast cancer cells to other lymph nodes but not the sentinel and axillary lymph nodes - a finding supported in cellular invasion assays using HLCS knockdown cells. Furthermore, overexpression of HLCS reduced survival time of patients with breast cancer. CONCLUSION: HLCS appears to be a prognostic marker for patients with breast cancer.

c-Myc directly targets an over-expression of pyruvate carboxylase in highly invasive breast cancer.

Here we showed that the c-Myc oncogene is responsible for overexpression of pyruvate carboxylase (PC) in highly invasive MDA-MB-231 cells. Pharmacological inhibition of c-Myc activity with 10074-G5 compound, resulted in a marked reduction of PC mRNA and protein, concomitant with reduced cell growth, migration and invasion. This growth inhibition but not migration and invasion can be partly restored by overexpression of PC, indicating that PC is a c-Myc-regulated pro-proliferating enzyme. Analysis of chromatin immunoprecipitation sequencing of c-Myc bound promoters revealed that c-Myc binds to two canonical c-Myc binding sites, locating at nucleotides -417 to -407 and -301 to -291 in the P2 promoter of human PC gene. Mutation of either c-Myc binding site in the P2 promoter-luciferase construct resulted in 50-60% decrease in luciferase activity while double mutation of c-Myc binding sites further decreased the luciferase activity in MDA-MB-231 cells. Overexpression of c-Myc in HEK293T cells that have no endogenous c-Myc resulted in 250-fold increase in luciferase activity. Mutation of either E-boxes lowered luciferase activity by 50% and 25%, respectively while double mutation of both sites abolished the c-Myc transactivation response. An electrophoretic mobility shift assay using nuclear proteins from MDA-MB-231 confirmed binding of c-Myc to both c-Myc binding sites in the P2 promoter. Bioinformatic analysis of publicly available transcriptomes from the cancer genome atlas (TCGA) dataset revealed an association between expression of c-Myc and PC in primary breast, as well as in lung and colon cancer tissues, suggesting that overexpression of PC is deregulated by c-Myc in these cancers.

Coordination of the Cell Wall Integrity and High-Osmolarity Glycerol Pathways in Response to Ethanol Stress in Saccharomyces cerevisiae.

During fermentation, a high ethanol concentration is a major stress that influences the vitality and viability of yeast cells, which in turn leads to a termination of the fermentation process. In this study, we show that the BCK1 and SLT2 genes encoding mitogen-activated protein kinase kinase kinase (MAPKKK) and mitogen-activated protein kinase (MAPK) of the cell wall integrity (CWI) pathway, respectively, are essential for ethanol tolerance, suggesting that the CWI pathway is involved in the response to ethanol-induced cell wall stress. Upon ethanol exposure, the CWI pathway induces the expression of specific cell wall-remodeling genes, including FKS2, CRH1, and PIR3 (encoding ß-1,3-glucan synthase, chitin transglycosylase, and O-glycosylated cell wall protein, respectively), which eventually leads to the remodeling of the cell wall structure. Our results revealed that in response to ethanol stress, the high-osmolarity glycerol (HOG) pathway plays a collaborative role with the CWI pathway in inducing cell wall remodeling via the upregulation of specific cell wall biosynthesis genes such as the CRH1 gene. Furthermore, the substantial expression of CWI-responsive genes is also triggered by external hyperosmolarity, suggesting that the adaptive changes in the cell wall are crucial for protecting yeast cells against not only cell wall stress but also osmotic stress. On the other hand, the cell wall stress-inducing agent calcofluor white has no effect on promoting the expression of GPD1, a major target gene of the HOG pathway. Collectively, these findings suggest that during ethanol stress, the CWI and HOG pathways collaboratively regulate the transcription of specific cell wall biosynthesis genes, thereby leading to adaptive changes in the cell wall.IMPORTANCE The budding yeast Saccharomyces cerevisiae has been widely used in industrial fermentations, including the production of alcoholic beverages and bioethanol. During fermentation, an increased ethanol concentration is the main stress that affects yeast metabolism and inhibits ethanol production. This work presents evidence that in response to ethanol stress, both CWI and HOG pathways cooperate to control the expression of cell wall-remodeling genes in order to build the adaptive strength of the cell wall. These findings will contribute to a better understanding of the molecular mechanisms underlying adaptive responses and tolerance of yeast to ethanol stress, which is essential for successful engineering of yeast strains for improved ethanol tolerance.

High Performance of Photosynthesis and Osmotic Adjustment Are Associated With Salt Tolerance Ability in Rice Carrying Drought Tolerance QTL: Physiological and Co-expression Network Analysis.

Understanding specific biological processes involving in salt tolerance mechanisms is important for improving traits conferring tolerance to salinity, one of the most important abiotic stresses in plants. Under drought and salinity stresses, plants share overlapping responsive mechanisms such as physiological changes and activation of signaling molecules, which induce and transmit signals through regulator genes in a regulatory network. In this study, two near isogenic lines of rice carrying chromosome segments of drought tolerance QTL on chromosome 8 from IR68586-F2-CA-31 (DH103) in the genetic background of sensitive cultivar "Khao Dawk Mali 105; KDML105" (designated as CSSL8-94 and CSSL8-95) were used to investigate physiological responses to salt stress [namely growth, Na+/K+ ratio, water status, osmotic adjustment, photosynthetic parameters, electrolyte leakage (EL), malondialdehyde (MDA), proline and sugar accumulations], compared with the standard salt tolerant (Pokkali; PK) and their recurrent parent (KDML105) rice cultivars. Physiological examination indicated that both CSSLs showed superior salt-tolerant level to KDML105. Our results suggested that salt tolerance ability of these CSSL lines may be resulted from high performance photosynthesis, better osmotic adjustment, and less oxidative stress damage under salt conditions. Moreover, to explore new candidate genes that might take part in salt tolerance mechanisms, we performed co-expression network analysis for genes identified in the CSSL rice, and found that Os08g419090, the gene involved with tetrapyrrole and porphyrin biosynthetic process (chlorophyll biosynthetic process), Os08g43230 and Os08g43440 (encoded TraB family protein and cytochrome P450, respectively) might have unprecedented roles in salt stress tolerance.